1. Field of the Invention
The present invention provides improved methods for nucleic acid detection. The novel methods for simultaneous nucleic acid amplification and detection enhance the speed and accuracy of prior detection methods and eliminate the need for sample processing following amplification. In a preferred embodiment, the method provides a modification of the polymerase chain reaction and utilizes agents whose fluorescence is enhanced upon binding double-stranded DNA. The methods provided herein have numerous applications, particularly in the fields of molecular biology, medical diagnostics and forensic sciences.
2. Description of Related Art
The disclosed nucleic acid detection methods offer the advantages of speed and simplicity over prior methods for detecting amplified nucleic acids. Nucleic acid detection techniques in general are particularly useful in medical diagnostic assays. For example, Falkow et al., U.S. Pat. No. 4,358,535 disclose a method for detecting pathogens by spotting a sample (e.g., blood, cells, saliva, etc.) on a filter, lysing the cells and fixing the DNA through chemical denaturation and heating. Then, labeled DNA probes are added and allowed to hybridize with the fixed sample DNA. Hybridization indicates the presence of the pathogen's DNA.
Nucleic acid detection using oligonucleotide probes has become a standard method for specific target detection. Numerous modifications of the method have been described, including culturing the target cells or organisms in situ on the filter, increasing the amount of target nucleic acid available for detection. Generally, these methods require that the DNA sample is noncovalently bound onto a solid support such as nitrocellulose or nylon and then hybridized to a labeled target-specific probe.
The sensitivity and specificity of nucleic acid detection methods was greatly improved by the invention of the polymerase chain reaction (PCR). PCR is a process for amplifying nucleic acids and involves the use of two oligonucleotide primers, an agent for polymerization, a target nucleic acid template, and successive cycles of denaturation of nucleic acid and annealing and extension of the primers to produce a large number of copies of a particular nucleic acid segment. With this method, segments of single copy genomic DNA can be amplified more than 10 million fold with very high specificity and fidelity. PCR methods are disclosed in U.S. Pat. No. 4,683,202, which is incorporated herein by reference.
Methods for detecting PCR products are particularly described in U.S. Pat. No. 4,683,195, which is incorporated herein by reference. Those methods require an oligonucleotide probe capable of hybridizing with the amplified target nucleic acid. European Patent Publication No. 237,362, which is incorporated herein by reference, also describes a PCR-based detection method termed "reverse dot blot", in which the probe, instead of the amplified DNA, is fixed to the membrane. According to the method, the target, rather than the probe, is labeled for hybridization. These methods require separate steps of amplification, capture, and detection and generally require several hours to complete. In the reverse dot-blot method, storage-stable target-specific reagents are preferred.
Alterative methods for detecting amplified nucleic acids are described in copending U.S. Ser. No. 076,394, filed Jul. 22, 1987, which is incorporated herein by reference. U.S. Ser. No. 076,394 describes PCR-based methods for simultaneous amplification and labeling of a target nucleic acid. The methods require that at least one amplification primer is labeled. The amplification primer can be labeled with, for example, a radioisotope for direct detection of the amplified product or labeled with a reagent suitable for capturing the product onto a solid support for subsequent detection.
Other means of detection include the use of fragment length polymorphism hybridization, allele-specific oligonucleotide (ASO) probes (Saiki et al., 1986, Nature 324:163), or direct sequencing via the dideoxy method using amplified DNA rather than cloned DNA. The fragment length polymorphism method detects insertions and deletions between PCR primers resulting in PCR products of different lengths, detectable by sizing. ASO methods are useful for detecting allelic sequence variations. In an example of ASO hybridization, the amplified DNA is fixed to a nylon filter (by, for example, UV irradiation) in a series of "dot blots," then allowed to hybridize with an oligonucleotide probe under stringent conditions. This method is also described in copending U.S. Ser. No. 347,495, filed May 4, 1989, which is incorporated herein by reference. The probe may be labeled with, for example, horseradish peroxidase (HRP) and detected by the presence of a blue precipitate following treatment with suitable oxidation reagents.
Copending U.S. Ser. No. 563,758, filed Aug. 6, 1990, and incorporated herein by reference, describes an alterative assay method for detecting amplified nucleic acids. The process employs the 5' to 3' nuclease activity of a nucleic acid polymerase to cleave annealed, labeled oligonucleotides from hybridized duplexes and release labeled oligonucleotide fragments for detection. The method is suitable for detecting PCR products and requires a primer pair and a labeled oligonucleotide probe having a blocked 3'-OH terminus to prevent extension by the polymerase.
Due to the enormous amplification possible with the PCR process, small levels of DNA carryover from samples with high DNA levels, positive control templates, or from previous amplifications, can result in PCR product even in the absence of purposefully added template DNA. Higuchi and Kwok, (1989, Nature 339:237-238 and Kwok) and Orrego, (in Innis et al., 1990, PCR Protocols: A Guide to Methods and Applications Academic Press, Inc., San Diego, Calif.), describe particular methods and precautions for practicing PCR with a minimum of cross contamination. U.S. Ser. No. 609,157, filed Nov. 2, 1990, describes improved methods for reducing the effects of cross contamination by the introduction of unconventional nucleotide bases. These references are incorporated herein by reference. Because the possibility of introducing contaminating DNA to a sample will be increased as the amount of handling steps required for sample preparation, processing, and analysis is increased, it would be preferable to minimize sample handling, particularly after the amplification reaction is complete.
A number of agents have been described for labeling nucleic acids, whether probe or target, for facilitating detection of target nucleic acid. Suitable labels may provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism or enzymatic activity and include, for example, fluorophores, chromophores, radioactive isotopes (particularly .sup.32 P and .sup.125 I) electron-dense reagents, enzymes, and ligands having specific binding partners.
Labeling is achieved by a number of means, such as chemical modification of a primer or probe to incorporate a label or the use of polymerizing agents to incorporate a modified nucleoside triphosphate into an extension product. Intercalating agents non-covalently bind the stacked bases of nucleic acids and as a result the fluorescence of the agent either increases or shifts to a different wavelength. For example, U.S. Pat. No. 4,582,789 describes several intercalating moieties including psoralens. Copending U.S. Ser. No. 076,394 describes methods for amplifying and detecting nucleic acids using psoralen labeled primers. Both the '789 patent and the '394 application are incorporated herein by reference.
Fluorescent dyes are suitable for detecting nucleic acids. For example, ethidium bromide is an intercalating agent that displays increased fluorescence when bound to double-stranded DNA rather than when in free solution (Sharp et al, 1973, Biochemistry 12:3055). Ethidium bromide can be used to detect both single- and double-stranded nucleic acids, although the affinity of ethidium bromide for single-stranded nucleic acid is relatively low. Ethidium bromide is routinely used to detect nucleic acids following gel electrophoresis. Following size fractionation on an appropriate gel matrix, for example, agarose or acrylamide, the gel is soaked in a dilute solution of ethidium bromide. The DNA is then visualized by examining the gel under UV light (see Maniatis et al., 1982 eds., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory.)
Alternative fluorescence based methods for detecting DNA have been described. For example, Morrison et al., 1989, Anal. Biochem., 183:231-244, which is incorporated herein by reference, describe a two probe method for detecting target DNA. One probe is labeled with fluorescein, the other probe, complementary to the first, is labeled with a quencher for fluorescein emission. The probes are allowed to anneal with denatured DNA containing the target sequence, and the amount of fluorescence is determined. Fluorescence increases to the extent that the fluorescein probe binds to unlabeled, complementary DNA rather than the complementary, quenching probe.
Mabuchi et al., 1990, Nucl. Acids Res. 18(24):7461-7462, which is incorporated herein by reference, describe a method for detecting DNA fragments based on the AT content of the nucleic acid segment. Two fluorochromes are used to stain size fractionated DNA in an agarose gel. The selective binding properties of different fluorochromes for AT rich regions are used to distinguish electrophoresed DNA fragments.
U.S. Pat. No. 4,257,774, which is incorporated herein by reference, describes the direct binding of fluorescent intercalators to DNA, e.g., ethidium salts, daunomycin, mepacrine and acridine orange, as well as 4'6-diamidino-.alpha.-phenylindole to quantitate the DNA. Fluorescence polarization is used for characterization of non-fluorescent DNA binding compounds which complete with the DNA binding dyes.
Oser and Valet (1990, Angew. Chem. Int. Engl. 29(10): 1167) describe a nucleic acid detection scheme that requires two oligonucleotide probes complementary to adjacent sites on a target. The probes are labeled differentially with either a salicylate or a DTPA ligand bearing a fluorescence emitter, Tb.sup.III. Hybridization of both probes to the target provides steric proximity of the two labels resulting in a measurable increase in Tb.sup.III fluorescence. The modified probes are prepared specifically for each target to be detected.
European Patent Publication No. 070,685 describes the use of fluorescent labeled polynucleotide probes in polynucleotide hybridization assays. According to the method, probes are prepared by attaching particular absorber-emitter moieties to the 3' and 5' ends of nucleic acid fragments. The fragments are capable of hybridizing to adjacent positions on a target DNA, so that, if both fragments are hybridized, the proximity of the absorber and emitter moieties results in detectable emitter fluorescence.
According to these methods, the fluorescent dye is introduced to the target DNA after all in vitro nucleic acid polymerization reactions have been completed. The inhibitory effects of intercalating agents on nucleic acid polymerases have been described in numerous publications (see for example, Kornberg, 1974, DNA Synthesis, W. H. Freman and Co., San Francisco, and Richardson, 1973, J. Mol. Biol. 78:703-714, which is incorporated herein by reference).
DNA binding dyes are useful as antibiotics because of the inhibitory effects on nucleic acid replication processes that result from the agent binding to the template. European Patent Publication No. 169,787 describes the use of intercalating agents for blocking replication of influenza or herpes virus. Kornberg (supra) describes a number of DNA binding agents, both intercalators and non-intercalators, and describes how each compound inhibits nucleic acid replication. At page 227, Kornberg specifically describes that ethidium bromide inhibits DNA replication.
A method for simultaneous amplification and detection of target nucleic acids would provide advantages over prior detection methods. Such a method would minimize the problems of sample contamination inherent in any process involving a series of manipulative steps for discerning a positive or negative test result. By eliminating sample handling and processing steps, a method for simultaneous amplification and detection of target nucleic acids would increase the speed and accuracy of current diagnostic methods. The present invention addresses and solves these needs.